Yersinia pestis was the pathogen responsible for the bubonic plague, also known as the Black Death. Although plague has long been considered a once-vanquished disease, the recent outbreak of the pneumonic plague caused by Yersinia pestis in Surat, India, proves that its biological potential can be expressed under appropriate environmental conditions. In addition, there is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror, as well as instruments of warfare for mass destruction. Thus, there is an urgent need to devise effective protective strategies that could be implemented soon after a bioterrorist attack. Although an understanding of the pathogenic processes induced by Yersinia pestis is incomplete, several of the bacterial virulence factors have been located on a naturally occurring 70 kb plasmid. The expression of a set of proteins encoded on the virulence plasmid known as YOPs (for Yersinia outer membrane proteins) is correlated with the capacity of the bacterium to avoid host defense mechanisms. One of the YOP proteins, YopH, is a protein tyrosine phosphatase (PTPase). The yopH gene is obligatory for pathogenesis, and plasmids that have a nonfunctional yopH gene are avirulent, suggesting that the Yersinia PTPase activity is essential for bacterial pathogenicity. Thus, specific inhibitors of the Yersinia PTPase are expected to be powerful agents to prevent and terminate the dissemination of Yersinia pestis infection. A multidisciplinary research program is designed to develop and evaluate specific inhibitors of the PTPase (YopH) from Yersinia pestis as novel anti-plague agents. Efficient and specific YopH substrates will be identified using detailed kinetic analysis of the PTPase active site specificity using libraries of aryl phosphates. Potent and selective YopH inhibitors will be prepared by converting the efficient and specific aryl phosphate substrates to their nonhydrolyzable difluorophosphonate derivatives. The in vivo efficacy of selected inhibitors will be assessed in a murine macrophage-like cell line, J774A.1, by measuring changes in endogenous levels of host tyrosine phosphorylation associated with the binding and phagocytosis of Yersinia pseudotuberculosis. The structural basis of inhibition specificity will be revealed by the determination of the three-dimensional structures of YopH complexed with high affinity inhibitors, which should provide framework for further improvement in inhibitor potency and specificity.